Project summary. This proposal describes a multi-disciplinary research program that aims to develop, validate and disseminate microfluidic technology to allow development of a live Drosophila embryo to be controlled in space and time using temperature steps. The process of Drosophila embryo development is robust - it works precisely even under varying environmental conditions such as temperature. While the function of many individual molecules present in Drosophila development is known, it is unknown how these molecules work together to make developmental network robust. New microfluidic technology that can differentially control temperature around different parts a living embryo could become a powerful tool in determining the mechanisms responsible for this robustness of development. Specific Aim 1focuses on development of new microfluidic technology that will use laminar flow to create a sharp temperature step around the embryo, where one part of the embryo will develop at the warmer temperature of one laminar stream, and the other part of the embryo will develop at the cooler temperature of the second laminar stream. The temperature profile at the surface of the embryo will be quantitatively characterized using numerical simulations and confocal microscopy. Real-time imaging of an embryo being exposed to temperature step is critical in identifying dynamic processes such as changes in protein concentration as a function of time. Specific Aim 2 adapts technology developed in Specific Aim 1to DIG and 2-photon microscopy in order to image embryonic development in the temperature step in real time. Proposed research in Specific Aim 3 will validate the microfluidic technology developed in Specific Aim 1and Specific Aim 2 by answering four important questions concerning the mechanism of robustness, both at the molecular level and at the level of nuclear divisions. Relevance: Understanding development is essential for understanding of human diseases and conditions caused by defects and errors in developmental and differentiation pathways (such as cancer and aging). Studying development in model organisms[unreadable]in particular, the fruit fly Drosophila melanogaster[unreadable]leads to a better understanding of human development, since many parts of the basic machinery of development are similar among organisms. The microfluidic technology developed in this proposal will enable understanding of the mechanism that provides error-free operation of developmental network in the fruit fly Drosophila melanogaster, and this technology will be extendable to testing and understanding errors in development of other model organisms